This disclosure relates generally to detection and characterization of nucleic acids. More specifically this disclosure relates to determining the sequences of nucleic acids using, for example, sequencing-by-synthesis techniques.
Our genome provides a blue print for predicting many of our inherent predispositions such as our likes and dislikes, talents, susceptibility to disease and responsiveness to therapeutic drugs. The human genome contains a sequence of over 3 billion nucleotides and it is the differences in just a fraction of those nucleotides that impart many of our unique characteristics. The research community is making impressive strides in unraveling the features that make up the blue print and with that a more complete understanding of how the information in each blue print relates to human health. However, a more complete understanding of how the information in each blue print relates to the living structures they encode, will require that tens-of-thousands or millions of genomes be sequenced. Only then will scientists be able to correlate the complexities of the genetic code with the variety of human characteristics. Furthermore, beyond the research effort the costs must come down in order to usher in the day when each person will have a copy of their own personal genome so that they can sit down with their doctor to determine appropriate choices for a healthy lifestyle or a proper course of treatment.
The most prevalent method for reading the sequence of nucleotides in a genome is sequencing-by-synthesis (SBS). In a typical SBS protocol, millions of genome fragments are attached to individual locations on the surface of a chip or microscope slide. The surface-attached fragments are subjected to repeated cycles of reagent delivery and detection such that the nucleotides in each fragment are ‘read’ one by one. The repeated cycles amount to a synthesis process in which each fragment acts as a template for synthesis of a complementary polynucleotide, one nucleotide per cycle. Each cycle includes (1) delivering blocked, labeled nucleotides (e.g., A, T, G, C) and a polymerase under conditions where the polymerase adds a single nucleotide to the growing complementary polynucleotide; (2) washing the surface to remove excess monomeric nucleotides that did not react; (3) detecting a label that was recruited to each fragment by the addition of the respective nucleotide to the complementary polynucleotide; (4) delivering a deblocking agent that removes a blocking group that was also recruited to each fragment by the addition of the respective nucleotide to the complementary polynucleotide; and (5) washing the surface to remove the deblocking agent from the surface so that it will not interfere with the next nucleotide addition step in the next cycle. Several cycles of reagent delivery and detection can be repeated to determine the sequences of the genomic DNA fragments.
In the above SBS protocol the blocking group prevents more than one nucleotide from being added to the growing complementary polynucleotide during each cycle. The blocking group is recruited to the complementary polynucleotide when the single nucleotide is added and a subsequent nucleotide cannot be added until the deblocking agent removes it. In each cycle of the SBS protocol, the deblocking agent is introduced to the surface bound fragments after the nucleotide monomers and polymerase have been washed away and after the detection step. Furthermore, extensive washing of the surface is carried out after the deblocking step. As such, the deblocking agent(s) are not present with the reagents used for nucleotide addition. This separation of deblocking agents from extension reagents provides for the incremental control of the SBS reaction whereby only a single nucleotide is added to a complementary polynucleotide during each cycle. The incremental control in turn provides for accuracy in detecting each nucleotide addition individually and in proper order.
Sequencing-by-synthesis has been a very successful methodology, but is still relatively expensive. A variety of approaches can be taken to address this problem. For example, the efficiency of manufacturing SBS reagents can be improved. Indeed, this approach has been taken by commercial suppliers of SBS platforms and has achieved incremental improvements. Another approach is miniaturization, for example, by increasing the density of fragments on detection surfaces or reducing reagent volumes used in fluidic steps. Several techniques have been devised that remove fluidic steps. For example, SBS has been carried out using nucleotides that do not have blocking moieties, thereby avoiding the need for a separate deblocking step and deblocking agents. Other techniques have been developed that avoid the need for nucleotides having label moieties and/or optical detection. However, many of the above approaches have inherent limitations that have to date limited their effectiveness at bringing down the cost of sequencing and that may ultimately prevent them from doing so.
What is needed is a reduction in the cost of sequencing that drives large genetic correlation studies carried out by research scientists and that makes sequencing accessible in the clinical environment for the treatment of individual patients making life changing decisions. The invention set forth herein satisfies this need and provides other advantages as well.